The role of membrane lipid microdomains (rafts) in FcgammaRIIA effector functions

Immunoglobulin G (IgG) dependent effector activity is an important factor in host defense and autoimmune diseases. Both macrophages and neutrophils express a family of Fcgamma receptors (FcgammaR) which recognize both monomeric and complexed IgG. One member of this family, FcgammaRIIA, is a transmembrane glycoprotein which mediates binding and internalization of large IgG-coated targets (phagocytosis) and small IgG-containing complexes (IC) (endocytosis). Phagocytosis differs from endocytosis in the requirement for actin versus clathrin and the dependency on specific kinases and phosphatases. FcgammaRIIA is known to translocate into lipids rafts upon binding IgG-containing targets.;We hypothesize that lipid rafts participate to different extents in the initial binding and internalization of IgG containing complexes by leukocytes. Observations using FcgammaRIIA transfected CHO cells indicate that disruption of lipid rafts with 8mM methyl-beta-cyclodextrin (MbetaCD) nearly abolishes binding of large IgG-coated 4.5mum polystyrene beads (26% binding versus untreated control). Furthermore, disruption of lipid rafts with 8mM MbetaCD subsequent to binding inhibited phagocytosis of these targets by 74%. Interestingly, identical experiments with 1.5mum beads displayed a similar loss of binding to their larger counterparts following MbetaCD treatment (34% of control), though no significant loss in internalization. The binding and internalization of small heat-aggregated IgG displayed significant, though muted losses in comparison to the large targets (binding 82% of control, internalization 74% of control). Finally, FRAP experiments were conducted to examine the role of lipid rafts in FcgammaRIIA mobility in the membrane. Results indicate that while there is not a significant effect on normal movement through the membrane, upon ligation, there is a significant lipid raft-associated effect on both the rate of movement and fraction of mobile FcgammaRIIA in the membrane (33% loss in recovery rate, 2.5-fold increase in immobile fraction).;Our current observations suggest that differences between phagocytosis and endocytosis may arise as early as the initial stages of ligand recognition. This is the first observance of a size-related dependency on lipid rafts for FcgammaRIIA function, and may signal an important avenue of research in both the understanding of the mechanisms involved and the potential for specific modulation of the Ab-dependent immune and inflammatory mechanisms.